Construction and Studies of Recombinant Mouse Prion Protein Expression System

普昂蛋白質 (prion protein, PrP) 是一個藉 GPI-anchor 連接在膜上的醣蛋白質，由正常構形 (PrPC) 轉變為致病構形 (PrPSc) 的過程，被廣泛接受為引起普昂疾病的原因，但這個轉變過程仍不清楚，包括醣基化的影響以及物種屏障 (species barrier) 的成因，都未成定論。我們試著利用可進行轉譯後修飾作用的表現系統，例如進行醣基化，來表現重組 PrP。因此，本論文在 Escherichia coli 與 Pichia pastoris 中建立了重組小鼠 PrP 的表現系統。將 E. coli 中純化得到的重組蛋白質進行澱粉樣蛋白質纖維之培養。以 thioflavin T (ThT) 呈色法監控纖維的形成，再藉由穿透式電子顯微鏡 (Transmission electron microscope, TEM) 觀察纖維的形狀。基於合成胜肽系統推導出關於物種屏障的理論，而建構出三個突變蛋白質。依照金倉鼠 PrP 的序列，將小鼠重組 PrP 序列中 108, 111, 138 這三個位置的胺基酸換成 methionine。重組小鼠 PrP(23-230) 蛋白質的表現系統也同時建構在 P. pastoris 中，結果顯示由酵母菌產生的重組 PrP 也會存在不可溶的蛋白質中。Prion protein (PrP) is a glycoprotein attached to plasma membrane via GPI-anchor. It is believed that the conversion from normal cellular PrP (PrPC) to pathogenic scrapie PrP (PrPSc) results in prion diseases. However, many issues still remained elusive, e.g. the effects of glycosylation and the factors that contributed to species barrier. We tried to express recombinant PrP in an expression system which was able to provide post-translational modifications, such as glycosylation. Therefore, recombinant wild type mouse PrP expression in Escherichia coli and Pichia pastoris were established. Recombinant wild type PrP purified from E. coli was used to form amyloid fibrils. The formation of the amyloid fibril was monitored by thioflavin T (ThT) assay. Transmission electron microscopy (TEM) was used to examine the morphology of the fibrils. Based on the theory for species barrier derived from synthetic peptide system established in previous study, three PrP(23-230) mutants were generated in an attempt to study the theory. The amino acids at positions of 108, 111, 138 in mouse PrP sequence were altered to methionine according to the PrP sequence of golden Syrian hamster. Recombinant mouse PrP(23-230) was also constructed for the expression in P. pastoris. It appeared that the recombinant PrP produced by yeast was also accumulated in the insoluble fraction.目錄................................................................................................................................…………….......I 縮寫表.................................................................................................................................................IV 摘要.......................................................................................................................................................V Abstract..............................................................................................................................................VI 第一章 緒論 1.1 普昂疾病的簡介 ......................................................................................................................1 1.1.1 羊搔癢症 (scrapie) ...................................................................................................1 1.1.2 庫賈氏症 (CJD, Creutzfeldt-Jakob disease) .....................................................2 1.1.3 古魯症 (kuru) .............................................................................................................3 1.1.4 致命性家族失眠症 (FFI, fatal familial insomnia) ...........................................4 1.1.5 狂牛症 (BSE, bovine spongiform encephalopathy, mad cow disease) 與 vCJD (variant CJD) ..............................................................................................4 1.2 普昂 (prion) 簡介與其近代研究 ......................................................................................6 1.3 普昂蛋白質之簡介 .................................................................................................................8 1.3.1 普昂蛋白質的發現 ...................................................................................................8 1.3.2 轉譯出普昂蛋白質的基因 .....................................................................................9 1.3.3 小鼠普昂蛋白質之序列與功能性區塊 ..............................................................9 1.3.4 普昂蛋白質的生理功能 ........................................................................................10 1.3.5 普昂蛋白質的結構 .................................................................................................11 1.4 普昂疾病的物種屏障 (species barrier) .........................................................................12 1.5 轉譯後修飾作用與普昂蛋白質轉變為致病形態的關係 .........................................13 1.5.1 醣基化對普昂蛋白質的影響 ..............................................................................14 1.5.2 GPI-anchor 對普昂蛋白質的影響 ...................................................................15 1.6 澱粉樣蛋白質 (amyloid) 與相關疾病之簡介 ...........................................................16 1.7 研究動機與方向 ...................................................................................................................17 第二章 材料與方法 2.1 實驗材料 ................................................................................................................................20 2.1.1 載體 ............................................................................................................................20 2.1.2 菌株 ............................................................................................................................21 2.1.2.1 大腸桿菌 ...............................................................................................21 2.1.2.2 酵母菌 ...................................................................................................22 2.2 實驗藥品 ................................................................................................................................22 2.2.1 一般化學藥品 ..........................................................................................................22 2.2.2 酵素 ............................................................................................................................22 2.2.3 培養基 ........................................................................................................................23 2.3 儀器設備 ................................................................................................................................24 2.4 實驗方法 ................................................................................................................................25 2.4.1 DNA 相關之操作方法 .........................................................................................25 2.4.1.1 質體 DNA 小量製備 .......................................................................25 2.4.1.2 質體 DNA 中量製備 .......................................................................26 2.4.1.3 DNA 限制酶切 (Restriction Enzyme Digestion) ....................27 2.4.1.4 洋菜膠體電泳 (Agarose Gel Electrophoresis) .........................27 2.4.1.5 DNA 片段純化 (DNA Gel Extraction) ........................................27 2.4.1.6 DNA 片段接合 (Ligation) ..............................................................28 2.4.1.7 大腸桿菌化學法轉型 ........................................................................28 2.4.1.8 酵母菌 Pichia pastoris 之電穿孔 (Electroporation) 轉型 28 2.4.1.9 酵母菌染色體 DNA 之抽取 .........................................................29 2.4.1.10 聚合酶鏈鎖反應 (Polymerase Chain Reaction, PCR) ...........30 2.4.1.11 聚合酶鏈鎖反應產物純化 (PCR clean up) ................................31 2.4.1.12 QuikChange® 點突變技術 .............................................................31 2.4.2 蛋白質相關之操作方法 ........................................................................................32 2.4.2.1 重組蛋白質之最佳表現條件篩選 .................................................32 2.4.2.2 重組蛋白質之表現 ............................................................................33 2.4.2.3 E. coli 表現系統中 inclusion bodies 之純化 .........................35 2.4.2.4 P. pastoris 表現系統中非可溶性蛋白質之純化 ......................36 2.4.2.5 重組蛋白質之純化 ............................................................................37 2.4.2.6 蛋白質電泳檢定 .................................................................................39 2.4.2.7 蛋白質免疫轉印法 ............................................................................42 2.4.2.8 澱粉樣蛋白質纖維養成 ...................................................................44 2.4.2.9 以 thioflavin T 法偵測澱粉樣蛋白質纖維之生成 .................44 2.4.2.10 穿透式電子顯微技術 (Transmission Electron Microscopy, TEM) 樣本之製備 .............................................................................45 第三章 結果與討論 3.1 重組小鼠普昂蛋白質在大腸桿菌表現系統中之研究 .............................................46 3.1.1 進行點突變使重組蛋白質與 C 端標記接合 ................................................46 3.1.2 最適表現條件之探討 ............................................................................................47 3.1.3 重組小鼠普昂蛋白質之 inclusion body 的分離與純化 ..........................47 3.1.4 重組小鼠普昂蛋白質的純化 ..............................................................................48 3.1.5 重組小鼠普昂蛋白質之澱粉樣蛋白質纖維養成 .........................................50 3.1.6 對重組小鼠普昂蛋白質進行點突變以模仿金倉鼠普昂蛋白質 ..............50 3.1.7 各突變株之表現與 inclusion bodies 的純化 ..............................................51 3.1.8 各突變株之蛋白質純化 ........................................................................................52 3.1.9 以突變蛋白質 rPrP_LVM 進行澱粉樣蛋白質纖維養成 ...........................53 3.2 重組小鼠普昂蛋白質在酵母菌表現系統中之研究 ..................................................55 3.2.1 小鼠普昂蛋白質之基因序列選殖 .....................................................................55 3.2.2 載體的轉型與高抗性轉型株之篩選 ................................................................57 3.2.3 最適表現菌株及最適表現條件之探討 ............................................................59 3.2.4 鑑別在 Pichia pastoris 中表現的重組蛋白質是否可溶 ..........................60 第四章 結論與未來展望 4.1 結論 .........................................................................................................................................62 4.2 未來展望 ................................................................................................................................63 參考文獻 .........................................................................................................................................64 圖與表 ..............................................................................................................................................72 碩士論文口試問答摘要 ...........................................................................................................10